Transgenic plant and the method for producing the same

ABSTRACT

The present invention is directed to a transgenic plant and a method for producing the same. In particular, the present invention is directed to a transgenic plant or a plant cell in which a nucleic acid molecule encoding an m6A demethylase is introduced, wherein said m6A demethylase has the following two domains: i) N-terminal domain (NTD) having the function of AlkB oxidation demethylase; and ii) C-terminal domain (CTD). The present invention is also directed to a method for producing said plant, comprising introducing a nucleic acid molecule encoding an m6A demethylase into a regenerable plant cell, and regenerating a transgenic plant from the regenerable plant cell.

TECHNICAL FIELD

The present invention is directed to a transgenic plant and a method forproducing the same. In particular, the present invention is directed tointroducing a nucleic acid molecule into a plant to increase the biomassand/or yield of the plant.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The Sequence Listing in an ASCII text file, named35034_SequenceListing.txt of 33.6 KB, created on May 24, 2017, andsubmitted to the United States Patent and Trademark Office via EFS-Web,is incorporated herein by reference.

BACKGROUND

Due to the increasing population in the world, the area of the availableearth for agriculture is decreasing. To increase the efficiency ofagriculture and increase the diversity of the horticultural plantsremains the main object of researches.

Conventional methods for improving crops and horticultural plantsutilize selective breeding technologies to identify plants with desiredproperties. This kind of selective breeding technologies, however, hasseveral defects: these technologies are generally labor intensive, andresult in plants which generally comprise heterogeneous geneticcomplements which do not always transfer desired traits from the patentplants.

The development of molecular biology allows people to manipulate animalsand plants. The genetic engineering of plants requires isolating andmanipulating genetic materials (generally in the form of DNA or RNA) andthen introducing genetic materials into plants. Such technology hasresulted in plants having a variety of improved economic, agriculturalor horticultural traits. A trait having special economic significance isa growth characteristic, for example, high yield.

Yield of seeds is a very important trait, for the seeds of many plantsare very important to the nutrition of human being and animals. Nomatter via the consumption of the seeds per se or that of the meatproducts based on processed seeds, crops such as maize, rice, wheat andsoybean, account for more than half of the total calories taken in byhuman. They are also origins of sugars, oils and many kinds ofmetabolites used for industrial processing. Based on the constant needof finding genes for increasing yield of seeds, the prior art hasdisclosed methods via manipulating hormone levels of plants(WO03/050287), via manipulating cell cycles (WO2005/061702) andmanipulating genes involved in salt stress reactions (WO 2004/058980).In addition to yield of seeds, size of a thousand seeds, weight of asingle plant, tiller number and/or plant height are also importanttraits for measuring yield of plants. Moreover, it should be clarifiedthat the growth rate of a plant is not necessarily related to its yield.For example, when the fertilizer is sufficient, rice tends to grow toofast and too high during the early stage and exhibits lodging during thelate stage. This leads to the decrease of yield.

Transgenic technologies introduce artificially isolated and modifiedgenes into the genome of an organism, and lead to hereditarymodifications of traits of the organism due to the expression of theintroduced genes. Researches of transgenic technologies of plants mostlyfocus on the fields of anti-insect genetic engineering, anti-diseasegenetic engineering, stress resistance genetic engineering, qualitygenetic engineering, etc. Transgenic plants which have beencommercialized are mainly anti-insect and anti-herbicide varieties. Theplanting of those varieties decreases the use of chemical pesticides by37%, increases the yield of crops by 22%, and increases the profit offarmers by 68%. The current transgenic technologies, however, increasethe yield indirectly by anti-insect and anti-herbicide properties, etc.

Previous researches on epigenetics generally focus on reversiblemodifications of DNA and histone. Recently, researchers gradually movetheir interest to the field of RNA modification. Up to now, scientistshave discovered hundreds of RNA modifications. N⁶-methyladenoesine (m⁶A)is the most abundant RNA modification in mRNA across all eukaryotes. Themodification of m⁶A has been discovered for more than forty years, butits function was not known until the inventor of the present applicationfound the demethylase of m⁶A, FTO protein for the first time (Jia et al,N⁶-methyladenosine in nuclear RNA is a major substrate of theobesity-associated FTO. Nat Chem Biol, 2011, 7 (12): 885-887), reportedthe reversibitly of RNA modification for the first time and started the“RNA epigenetics (or Epitranscriptome)” research in 2011. In 2012,researchers developed m⁶A antibody assisted whole transcriptome m⁶Ahigh-throughput sequencing technology, m⁶A-seq (or MeRIP). The result ofthe sequencing shows that there are about 12,000 m⁶A sites in human andmouse cells, mainly distribute on 7,000 mRNAs transcribed from encodinggenes and 300 non-coding RNAs (ncRNAs) transcribed from non-coding genes(Dominissini et al, Topology of the human and mouse m⁶A RNA methylomesrevealed by m⁶A-seq. Nature, 2012, 485 (7397): 201-206; Meyer et al,Comprehensive analysis of mRNA methylation reveals enrichment in 3′ UTRsand near stop codons. Cell, 2012, 149 (7): 1635-1646). Up to now, it hasbeen found in the mammalian that the main components of themethyltransferase are METTL3, METTL14 and WTAP (liu et al, AMETTL3-METTL14 complex mediates mammalian nuclear RNA N⁶-adenosinemethylation. Nat Chem Biol, 2014, 10 (2): 93-95; Ping et al, MammalianWTAP is a regulatory subunit of the RNA N6-methyladenosinemethyltransferase. Cell Res, 2014, 24 (2): 177-189). There are two kindsof m⁶A demethylases, which are FTO (fat mass and obesity associated) andALKBH5 (Jia et al, N6-methyladenosine in nuclear RNA is a majorsubstrate of the obesity-associated FTO. Nat Chem Biol, 2011, 7 (12):885-887: Zheng et al, ALKBH5 is a mammalian RNA demethylase that impactsRNA metabolism and mouse fertility. Mol Cell, 2013, 49 (1): 18-29). m⁶Aregulates the metabolic processing of mRNAs, including splicing, nuclearexport, stability and protein translation via m⁶A binding proteins.

In the plant field, researchers found that mRNAs of all plants comprisem⁶A extensively. It is produced by m⁶A modifying enzymes (currentlyreported modifying enzyme subunits are classified as MTA (METTL3homologous gene) and FIP37 (WTAP homologous gene)), and has importantregulatory effect on the growth and development of plants. Researchersfound that when MTA or FIP37 was knocked out from Arabidopsis thaliana,the seed could not germinated normally. When MTA or FIP37 was partiallycomplemented during the germinating stage, after germinating, the lossof m⁶A from Arabidopsis thaliana severely inhibited the normal growthand development of the plant (Zhong, S., Li, H., Bodi, Z., Button, J.,Vespa, L., Herzog, M., and Fray, R. G (2008). MTA is an Arabidopsismessenger RNA adenosine methylase and interacts with a homolog of asex-specific splicing factor. Plant Cell 20, 1278-1288; Shen, L., Liang,Z., Gu, X., Chen, Y., Teo, Z. W., Hou, X., Cai, W. M., Dedon, P. C.,Liu, L., and Yu, H. (2016). N⁶-Methyladenosine RNA ModificationRegulates Shoot Stem Cell Fate in Arabidopsis. Dev Cell 38, 186-200).

The inventor of the present application previously found that during thedemethylation process of m⁶A in RNA, FTO produced two relatively stablenew modifications, hm⁶A (N⁶-hydroxymethyladenosine) and f⁶A(N⁶-formyladenosine), which had potential regulatory effect on RNAprocessing (Fu et al, FTO-mediated formation ofN⁶-hydroxymethyladenosine and N⁶-formyladenosine in mammalian RNA. NatCommun, 2013, 4:1798). New experimental data of the inventor showed thatFTO could remove tRNA methylation modifications (the data has not beenpublished).

It has been the task of the researches of all agriculture scientiststhat how to increase the yield of crops in limited area of earth to feedincreasing population and how to directly and effectively increase thebiomass and yield of plants. For crops (such as rice, wheat, maize),traditional transgenic technologies and hybridization breedingtechnologies may optimize a certain single gene, and increase the yieldby 10%˜30%. To achieve extremely high yield, it needs synergetic effectsof many genes. The regulation of the metabolic level of mRNA by RNAmethylation modification, provides the possibility of a method forregulating a single gene to achieve high yield or increase biomass.

SUMMARY OF THE INVENTION

The inventor has surprisingly found that by introducing m⁶A demethylaseFTO into plants, the metabolic level of mRNA may be regulated, and itprovides the possibility of a method for regulating a single gene toachieve high yield and/or increase biomass. To efficiently increase theyield and/or biomass of plants, the inventor introduced a heterogenousm⁶A demethylase by transgenic technologies, and dynamically regulate thecontent of m⁶A in mRNAs of plants so as to regulate the splicing,nuclear export, stability and protein translation of mRNAs. Comparedwith the traditional hybridization breeding method which increases theyield by 20-30%, with the method of the present invention, the yield ofplants increased 4 folds and the biomass increased 4 folds by theregulation of a single gene. It really achieved the high yield and highbiomass of plants by the regulation of a single gene.

In particular, the present invention is directed to the followingaspects: In one aspect, the present invention is directed to atransgenic plant or plant cell in which a nucleic acid molecule encodingan m⁶A demethylase is introduced, wherein said m⁶A demethylase has thefollowing two domains:

i) N-terminal domain (NTD) having the function of AlkB oxidationdemethylase; and

ii) C-terminal domain (CTD).

In another aspect, the present invention is directed to a method forproducing a transgenic plant exhibiting an increased biomass, anincreased yield (for example, increased seed/grain yield, increasedtuber yield, increased leaf yield, increased stem yield, increased rootyield, increased seed cotton yield) or the combination thereof, whereinsaid method comprises:

a) introducing a nucleic acid molecule encoding an m⁶A demethylase intoa regenerable plant cell, wherein said m⁶A demethylase has the followingtwo domains:

-   -   i) N-terminal domain (NTD) having the function of AlkB oxidation        demethylase; and    -   ii) C-terminal domain (CTD); and

b) regenerating a transgenic plant from the regenerable plant cell,wherein the transgenic plant comprises in its genome said nucleic acidmolecule encoding the m⁶A demethylase, and exhibits an increasedbiomass, an increased yield or the combination thereof when comparedwith a control plant which does not comprise the nucleic acid moleculeencoding the m⁶A demethylase.

In one embodiment, said method further comprises:

c) obtaining a progeny plant derived from the transgenic plant of stepb), wherein said progeny plant comprises in its genome said nucleic acidmolecule encoding the m⁶A demethylase, and exhibits an increasedbiomass, an increased yield or the combination thereof when comparedwith a control plant which does not comprise the nucleic acid moleculeencoding the m⁶A demethylase.

In one embodiment, the aforesaid m⁶A demethylase is a FTO protein. SaidFTO protein is from vertebrates or marine algae.

In one embodiment, said FTO protein has at least 40%, preferably atleast 50%, more preferably at least 60%, more preferably at least 70%,more preferably at least 80%, more preferably at least 90%, morepreferably at least 95%, more preferably at least 99%, most preferably100% identity to any one of SEQ ID NOs:1-4.

In one embodiment, said nucleic acid molecule encoding the m⁶Ademethylase has at least 90%, preferably at least 95%, more preferablyat least 99%, most preferably 100% identity to any one of SEQ IDNOs:5-12.

The transgenic plant of the present invention exhibits an increasedbiomass, an increased yield or the combination thereof when comparedwith a control plant which does not comprise the nucleic acid moleculeencoding the m⁶A demethylase.

In one embodiment, the plant of the present invention is selected fromthe group consisting of rice, maize (Zea mays), soybean, tobacco,potato, alfalfa (Medicago satliva), rape (Brassica), Russian dandelion(Taraxacum Kok-saghyz), cotton, wheat, millet (Panicum miliaceum), flax,sunflower and false flax (Camelina saliva) The present invention is alsodirected to a tissue, an organ, a pollen, a seed, a grain, a fruit and aprogeny plant of the aforesaid transgenic plant.

DESCRIPTION OF THE DRAWINGS AND THE SEQUENCES

FIG. 1 shows the map of pCAMBIA1307 plasmid in which a nucleic acidencoding FTO is introduced.

SEQ ID NO: 1 is the sequence of human (Homo sapiens) FTO protein.

SEQ ID NO: 2 is the sequence of pig (Sus scrofa) FTO protein.

SEQ ID NO: 3 is the sequence of cattle (Bos taurus) FTO protein.

SEQ ID NO: 4 is the sequence of Ostreococcus lucimarinus FTO protein,from a type of marine algae, Ostreococcus lucimarinusn.

SEQ ID NOs: 5 and 6 are nucleic acid sequences encoding human FTOprotein. SEQ ID NO:5 is the natural sequence isolated from human, andSEQ ID NO:6 is the sequence which has been codon-optimized for theexpression in plants.

SEQ ID NOs: 7 and 8 are nucleic acid sequences encoding pig FTO protein.SEQ ID NO: 7 is the natural sequence isolated from pig, and SEQ ID NO: 8is the sequence which has been codon-optimized for the expression inplants.

SEQ ID NOs: 9 and 10 are nucleic acid sequences encoding cattle FTOprotein. SEQ ID NO: 9 is the natural sequence isolated from cattle, andSEQ ID NO: 10 is the sequence which has been codon-optimized for theexpression in plants.

SEQ ID NOs: 11 and 12 are nucleic acid sequences encoding Ostreococcuslucimarinus FTO protein. SEQ ID NO: 11 is the natural sequence isolatedfrom Ostreococcus lucimarinus, and SEQ ID NO: 12 is the sequence whichhas been codon-optimized for the expression in plants.

DETAILED DESCRIPTION OF THE INVENTION

m⁶A demethylase or the homologs thereof and the nucleic acids encodingsaid demethylase or the homologs may be used to produce the transgenicplant of the present invention. The m⁶A demethylase used by the presentinvention may be present in any vertebrates and marine algae. Saidenzyme consists of nuclear localization sequence (NLS) and the followingtwo domains: i) N-terminal domain (NTD) having the function of AlkBoxidation demethylase; and ii) C-terminal domain (CTD).

As used herein “homolog” means a protein in a group of proteins thatperform the same biological function. Homologs are expressed byhomologous genes. Homologous genes include naturally occurring allelesand artificially-created variants. Degeneracy of the genetic codeprovides the possibility to substitute at least one base of the proteinencoding sequence of a gene with a different base without causing theamino acid sequence of the polypeptide produced from the gene to bechanged. Homologs are proteins that, when optimally aligned, have atleast 40% identity, more preferably about 50% or higher, more preferablyabout 60% or higher, more preferably about 70% or higher, morepreferably at least 80% and even more preferably at least 90% identityover the full length of a protein identified as increasing the yieldand/or biomass of plants when expressed in plant cells.

Homologs are be identified by comparison of amino acid sequence, e.g.manually or by use of a computer-based tool using known homology-basedsearch algorithms such as those commonly known and referred to as BLAST,FASTA, and Smith-Waterman. A local sequence alignment program, e.g.BLAST, can be used to search a database of sequences to find similarsequences, and the summary Expectation value (E-value) used to measurethe sequence base similarity. As a protein hit with the best E-value fora particular organism may not necessarily be an ortholog or the onlyortholog, a reciprocal query is used in the present invention to filterhit sequences with significant E-values for ortholog identification. Thereciprocal query entails search of the significant hits against adatabase of amino acid sequences from the base organism that are similarto the sequence of the query protein. A hit is a likely ortholog, whenthe reciprocal query's best hit is the query protein itself or a proteinencoded by a duplicated gene after speciation. A further aspect of theinvention comprises functional homolog proteins that differ in one ormore amino acids from those of disclosed protein as the result ofconservative amino acid substitutions, for example substitutions areamong: acidic (negatively charged) amino acids such as aspartic acid andglutamic acid; basic (positively charged) amino acids such as arginine,histidine, and lysine; neutral polar amino acids such as glycine,serine, threonine, cysteine, tyrosine, asparagine, and glutamine;neutral nonpolar (hydrophobic) amino acids such as alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan, and methionine;amino acids having aliphatic side chains such as glycine, alanine,valine, leucine, and isoleucine; amino acids having aliphatic-hydroxylside chains such as serine and threonine; amino acids havingamide-containing side chains such as asparagine and glutamine; aminoacids having aromatic side chains such as phenylalanine, tyrosine, andtryptophan; amino acids having basic side chains such as lysine,arginine, and histidine; amino acids having sulfur-containing sidechains such as cysteine and methionine; naturally conservative aminoacids such as valine-leucine, valine-isoleucine, phenylalanine-tyrosine,lysine-arginine, alanine-valine, aspartic acid-glutamic acid, andasparagine-glutamine. A further aspect of the homologs encoded by DNAuseful in the transgenic plants of the invention are those proteins thatdiffer from a disclosed protein as the result of deletion or insertionof one or more amino acids in a native sequence.

As used herein, “percent identity” means the extent to which twooptimally aligned DNA or protein segments are invariant throughout awindow of alignment of components, for example nucleotide sequence oramino acid sequence. An “identity fraction” for aligned segments of atest sequence and a reference sequence is the number of identicalcomponents that are shared by sequences of the two aligned segmentsdivided by the total number of sequence components in the referencesegment over a window of alignment which is the smaller of the full testsequence or the full reference sequence. “Percent identity” (“%identity”) is the identity fraction times 100.

The m⁶A demethylase used in the present invention may be SEQ ID NO: 1,2, 3 or 4 of the homolog thereof. Said homolog has at least 40%, 41%,42%, 43%, 44%, 45%, 46%, 47%, 48%, 4906, 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59, 60%, 61%, 62%, 63%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%0, or 99% sequence identity to any one of SEQ ID NOs: 1-4.

The nucleic acid encoding the m⁶A demethylase used in the presentinvention may be any one of SEQ ID NOs: 5-12 or the homologous genethereof. Said homologous gene has at least 40%, 41%, 42%, 43%, 44%, 45%,46%, 47%, 48%, 49%, 50%, 51%, 52%, 530, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to any one of SEQ ID NOs: 5-12.

The nucleic acid encoding the m⁶A demethylase defined herein may not bea full-length nucleic acid. A part of the nucleic acid encoding the m⁶Ademethylase defined herein may be prepared by making one or moredeletions from the full-length nucleic acid.

Another nucleic acid variant used in the present invention is a nucleicacid which hybridizes with the nucleic acid encoding the m⁶A demethylasedefined herein or with a part of the nucleic acid encoding the m⁶Ademethylase defined herein under stringent conditions.

Appropriate stringent conditions which promote DNA hybridization are,for example, 6.0× sodium chloride/sodium citrate (SSC) at about 45° C.,followed by a wash of 2.0×SSC at 5° C., are known to those skilled inthe art or can be found in Current Protocols in Molecular Biology(1989). For example, the salt concentration in the wash step can beselected from a low stringency of about 2.0×SSC at 50° C. to a highstringency of about 0.2×SSC at 50° C. In addition, the temperature inthe wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.Both temperature and salt may be varied, or either the temperature orthe salt concentration may be held constant while the other variable ischanged. The nucleic acid used in the present invention may specificallyhybridize with a nucleic acid encoding a FTO protein, for example, anyone of SEQ ID NOs: 5-12 or a part thereof under said conditions.

All or a portion of the nucleic acids of the present invention may besynthesized using codons preferred by a selected host. Species-preferredcodons may be determined, for example, from the codons used mostfrequently in the proteins expressed in a particular host species.Therefore, the nucleic acids of the present invention include thoseobtained by making codon-optimization to natural FTO proteins for theexpression in plants. Other modifications of the nucleotide sequencesmay result in mutants having slightly altered activity.

The present invention is directed to a transgenic plant in which a geneencoding the aforesaid m⁶A demethylase FTO or a homolog thereof isintroduced, or a progeny plant thereof. The present invention is alsodirected to a cell, a tissue, an organ, a pollen, a seed, a grain or afruit of said plant.

“Introduced” in the context of inserting a nucleic acid fragment (e.g.,a recombinant DNA construct) into a cell, means “transfection” or“transformation” or “transduction” and includes reference to theincorporation of a nucleic acid fragment into a eukaryotic orprokaryotic cell where the nucleic acid fragment may be incorporatedinto the genome of the cell (e.g., chromosome, plasmid, plastid ormitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA).

“Plant” includes reference to whole plants, plant organs, plant tissues,seeds and plant cells and progeny of same. Plant cells include, withoutlimitation, cells from seeds, suspension cultures, embryos, meristematicregions, callus tissue, leaves, roots, shoots, gametophytes,sporophytes, pollen, and microspores. “Progeny” comprises any subsequentgeneration of a plant.

According to the use, the plants of the present invention may be foodcrops, economic crops, vegetable crops, fruits, flowers, grasses, trees,industrial raw material crops, feed crops or medicine crops.Specifically, said food crops include rice, maize, soybean, beans, yams,potato, hulless barley, broad bean, wheat, barley, millet, rye, oat,sorghum, etc.; Said economic crops include oil tea, rape, rapeseed,flax, false flax (Camelina sativa), peanut, oil flax (Linumusitatissimum), mariguana (Cannabis sativa), sunflower, tobacco, cotton,beet, sugarcane, etc.; said vegetable crops include radish, Chinesecabbage, tomato, cucumber, hot pepper, carrot, etc.; said fruits includepear, apple, walnut, cherry, strawberry, jujube or peach; said flowersinclude flowers for view, for example, orchid, chrysanthemum, carnation,rose, green plants, etc., said grasses and trees include populus, heveabrasiliensis, taxus chinensis, and those for urban greening or thoseliving in deserts and harsh conditions such as drought; said industrialraw material crops include Russian dandelion, guayule, jatropha curcas,etc., said feed crops include the foodstuff for livestock, such asalfalfa etc.; said drug crops include ginseng, angelica and ganoderma,etc.

“Transgenic plant” includes reference to a plant which comprises withinits genome a heterologous polynucleotide. Preferably, the heterologouspolynucleotide is stably integrated within the genome such that thepolynucleotide is passed on to successive generations. The heterologouspolynucleotide may be integrated into the genome alone or as part of arecombinant DNA construct.

“Heterologous” with respect to sequence means a sequence that originatesfrom a foreign species, or, if from the same species, is substantiallymodified from its native form in composition and/or genomic locus bydeliberate human intervention.

The present invention has prepared a DNA construct which comprises anucleic acid molecule encoding the FTO protein described herein. Theconstruct may comprise the nucleic acid molecule encoding the FTOprotein described herein optionally operably linked to a promotersequence which functions in a host cell. Other construct components mayinclude additional regulatory elements, such as 5′ leaders and intronsfor enhancing transcription, 3′ untranslated regions (such aspolyadenylation signals and sites), DNA for transit, signal peptides, orone or more selective maker genes.

As used herein, “promoter” means regulatory DNA for initializingtranscription. A plant promoter is a promoter capable of initiatingtranscription in plant cells whether or not its origin is a plant cell,e.g. is it well known that Agrobacterium promoters are functional inplant cells. Thus, plant promoters include promoter DNA obtained fromplants, plant viruses and bacteria such as Agrobacterium andBradyrhizobium bacteria. Examples of promoters under developmentalcontrol include promoters that preferentially initiate transcription incertain tissues, such as leaves, roots, or seeds. Such promoters arereferred to as “tissue preferred”. Promoters that initiate transcriptiononly in certain tissues are referred to as “tissue specific”. A “celltype” specific promoter primarily drives expression in certain celltypes in one or more organs, for example, vascular cells in roots orleaves. An “inducible” or “repressible” promoter is a promoter which isunder environmental control. Examples of environmental conditions thatmay affect transcription by inducible promoters include anaerobicconditions, or certain chemicals, or the presence of light. Tissuespecific, tissue preferred, cell type specific, and inducible promotersconstitute the class of “non-constitutive” promoters. A “constitutive”promoter is a promoter which is active under most conditions. Promotersuseful in the present invention are not specifically limited. Thoseskilled in the art may select suitable promoters according to theirknowledge.

As used herein “operably linked” means the association of two or moreDNA fragments in a DNA construct so that the function of one, e.g.protein-encoding DNA, is controlled by the other, e.g. a promoter.

The DNA construct generally contain a selective maker gene. Selectivemarker genes are used to provide an efficient system for identificationof those cells that are stably transformed by receiving and integratinga transgenic DNA construct into their genomes. Preferred marker genesprovide selective markers which confer resistance to a selective agent,such as an antibiotic or herbicide. Potentially transformed cells areexposed to the selective agent. In the population of surviving cellswill be those cells where, generally, the resistance-conferring gene isintegrated and expressed at sufficient levels to permit cell survival.Cells may be tested further to confirm stable integration of theexogenous DNA. Commonly used selective marker genes include thoseconferring resistance to antibiotics such as kanamycin and paromomycin(nptII), hygromycin B (aph IV) and gentamycin (aac3 and aacC4) orresistance to herbicides such as glufosinate (bar or pat) and glyphosate(aroA or EPSPS). Examples of such selective markers are illustrated inU.S. Pat. Nos. 5,550,318; 5,633,435; 5,780,708 and 6,118,047, all ofwhich are incorporated herein by reference. Selective markers whichprovide an ability to visually identify transformants can also beemployed, for example, a gene expressing a colored or fluorescentprotein such as a luciferase or green fluorescent protein (GFP) or agene expressing a beta-glucuronidase or uidA gene (GUS) for whichvarious chromogenic substrates are known.

The introduction of the recombinant DNA construct into plants may becarried out by any suitable technique, including but not limited todirect DNA uptake, chemical treatment, electroporation, microinjection,cell fusion, infection, vector mediated DNA transfer, bombardment, orAgrobacterium mediated transformation. For Agrobacterium tumefaciensbased plant transformation system, additional elements present ontransformation constructs will include T-DNA left and right bordersequences to facilitate incorporation of the recombinant polynucleotideinto the plant genome.

In general it is useful to introduce recombinant DNA randomly, i.e. at anon-specific location, in the genome of a target plant line. In specialcases it may be useful to target recombinant DNA insertion in order toachieve site-specific integration, for example to replace an existinggene in the genome, to use an existing promoter in the plant genome, orto insert a recombinant polynucleotide at a predetermined site known tobe active for gene expression. Several site specific recombinationsystems exist which are known to function in plants include cre-lox asdisclosed in U.S. Pat. No. 4,959,317 and FLP-FRT as disclosed in U.S.Pat. No. 5,527,695, both incorporated herein by reference.

Transformation methods of this invention are preferably practiced intissue culture on media and in a controlled environment. “Media” refersto the numerous nutrient mixtures that are used to grow cells in vitro,that is, outside of the intact living organism. Recipient cell targetsinclude, but are not limited to, meristem cells, callus, immatureembryos and gametic cells such as microspores, pollen, sperm and eggcells. It is contemplated that any cell from which a fertile plant maybe regenerated is useful as a recipient cell. Callus may be initiatedfrom tissue sources including, but not limited to, immature embryos,seedling apical meristems, microspores and the like. Cells capable ofproliferating as callus are also recipient cells for genetictransformation. Practical transformation methods and materials formaking transgenic plants of this invention, for example various mediaand recipient target cells, transformation of immature embryo cells andsubsequent regeneration of fertile transgenic plants are disclosed inU.S. Pat. Nos. 6,194,636 and 6,232,526, which are incorporated herein byreference.

The development or regeneration of plants containing the foreign,exogenous isolated nucleic acid fragment that encodes a protein ofinterest is well known in the art. The regenerated plants areself-pollinated to provide homozygous transgenic plants. Otherwise,pollen obtained from the regenerated plants is crossed to seed-grownplants of agronomically important lines. Conversely, pollen from plantsof these important lines is used to pollinate regenerated plants. Atransgenic plant of the present invention containing the nucleic acidmolecule encoding the FTO protein is cultivated using methods well knownto one skilled in the art.

The seeds of transgenic plants can be harvested from fertile transgenicplants and be used to grow progeny generations of transformed plants ofthis invention including hybrid plants line for selection of plantshaving an enhanced trait. In addition to direct transformation of aplant with the nucleic acid molecule encoding the FTO protein,transgenic plants can be prepared by crossing a first plant having thenucleic acid molecule encoding the FTO protein with a second plantlacking the nucleic acid molecule. For example, the nucleic acidmolecule encoding the FTO protein can be introduced into first plantline that is amenable to transformation to produce a transgenic plantwhich can be crossed with a second plant line to introgress the nucleicacid molecule encoding the FTO protein into the second plant line. Thetransgenic plant derived from the plant cell of the present invention iscultivated to produce increased yield and/or biomass compared with acontrol plant. As used herein a “control plant” means a plant that doesnot contain the nucleic acid molecule encoding the FTO protein. Acontrol plant is to identify and select a transgenic plant that hasincreased yield and/or biomass. A suitable control plant can be anon-transgenic plant of the parental line used to generate a transgenicplant, i.e. devoid of the nucleic acid molecule encoding the FTOprotein. A suitable control plant may in some cases be a progeny of ahemizygous transgenic plant line that does not contain the nucleic acidmolecule encoding the FTO protein, known as a negative segregant.

The “yield” of the transgenic plant described herein means the harvestamount of the product desired by the cultivation. The standards forevaluating the yields of different plants are different. For example,the subject of the evaluation of the yields of cereal crops (rice,wheat, maize, etc.), beans and oil crops (soybean, peanut, rape, etc.)is the seed (grain); that of cotton is the seed cotton or the lintcotton; that of yam crops (sweet potato, potato, cassava, etc.) is thetuberous root or tuber; that of bast fiber crops is the fiber of stemsor the fiber of leaves; that of sugarcane is the stem; that of beet isthe root; that of tobacco is the leaf; that of green manure crops(alfalfa, trefoil, etc.) is the stem and leaf, etc. The meaning of theyield of the same plant differs when it is cultivated for differentpurposes. For example, when maize is cultivated as food and fine feedcrop, the yield is the harvest amount of grains, and when it iscultivated as silage, the yield includes the total harvest amount ofstems, leaves and ears. The increased yield of the transgenic plant ofthe present invention may be measured by many means, including measuringweight, seed number per plant, seed weight, tuber weight, seed numberper unit area (i.e. seeds, or weight of seeds, per acre), bushels peracre, tonnes per acre, tons per acre, kilo per hectare.

Those skilled in the art can determine the meaning of the yield for eachplant and the standard for evaluating it according to the knowledge inthe art.

Biomass means the total mass of the existing organic materials of anorganism. It is expressed as dry weight, fresh weight, tiller number,etc. in the present invention. The increased biomass of the transgenicplant of the present invention may be measured by many means, includingmeasuring weight, dry weight or fresh weight of the overground parts perplant, tiller number, dry weight of the overground parts per unit area(i.e. dry weight or fresh weight, per acre), bushels per acre, tonnesper acre, tons per acre, kilo per hectare.

EXAMPLES

The present invention is further illustrated in the following Examples.It should be understood that these Examples, while indicatingembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

Furthermore, various modifications of the invention in addition to thoseshown and described herein will be apparent to those skilled in the artfrom the foregoing description. Such modifications are also intended tofall within the scope of the appended claims.

The materials used in the following examples were as follows:

-   1. Formulation of YEP broth for the growth of Agrobacterium (per    liter): yeast extract 10 g/L+peptone 10 g/L+NaCl 5 g/L, PH 7.2. For    the solid medium, 15 g/L agar was added.-   2. Agrobacterium re-suspension AAM broth: 50 ml 20×AA macroelement,    10 ml 100×FeEDTA, 10 ml 100×B5 macroelement, 10 ml 100×B5 vitamin,    100 ml 10×AA amino acid, 1 ml 100 mM acetosyringone, 68.5 g sucrose,    36 g glucose, 0.5 g hydrolyzed casein. The volume was adjusted to    1000 ml. The pH was adjusted to 5.2. Sterilized with a 0.2 mm    cellulose acetate membrane.-   3. 20×AA macroelement: 59 g KCl, 3 g CaCl₂.2H₂O, 10 g MgSO₄.7H₂O and    3 g NaH₂PO₄.H₂O. The volume was adjusted with distilled water to    1 L. Stored at 4° C.-   4. O1×AA amino acid: 8.76 g Gin, 2.66 g Asp, 1.74 g Arg and 75 mg    Gly. The volume was adjusted with distilled water to 1 L. Sterilized    with a 0.2 mm cellulose acetate membrane. Stored at 4° C.-   5. 10 ml 100×B5 vitamin: 10 g myoinositol, 1 g thiamine    hydrochloride, 100 mg pyridoxine hydrochloride and 100 mg nicotinic    acid. The volume was adjusted with distilled water to 1 L. Stored at    4′C.-   6. 100×B5 macroelement: 1.320 mg MnSO₄.4H₂O, 200 mg ZnS₄.7H₂O, 2.5    mg CuSO₄.5H₂O, 25 mg Na₂MoO₄.2H₂O, 2.5 mg CoCl₂.6H₂O, 300 mg H₃BO₃    and 75 mg KI. The volume was adjusted with distilled water to 1 L.    Stored at 4° C.-   7. NB medium: macroelements and microelements of N6 medium, organic    elements of B5 medium, 300 mg/L hydrolyzed casein, 500 mg/L    glutamine, 30 g/L sucrose and 8 g/L agar.

Example 1: Obtaining cDNA of the m⁶A Demethylase Gene

The FTO genes were searched from the database of National Center forBiotechnology Information (NCBI). Amino acid sequences (SEQ ID NOs: 1-4)and nucleic acid sequences (SEQ ID NOs: 5, 7, 9 and 11) of human (Homosapiens) FTO, pig (Sus scrofa) FTO, cattle (Bos Taurus) FTO and alga(Ostreococcus lucimarinus CCE9901) FTO were obtained. The correspondingcDNAs were purchased, or synthesized by GenScript when there are nocommercialized cDNAs. The amino acid sequences of SEQ ID NOs: 1-4 wereoptimized to plant codons to synthesize the codon-optimized nucleic acidsequences SEQ ID NOs: 6, 8, 10 and 12.

The FTO gene cDNAs used in the following examples are SEQ ID NO:5, 7, 9and 11.

Example 2: Cloning the m⁶A Demethylase Gene

The FTO gene cDNAs (SEQ ID NO:5, 7, 9, 11) were cloned into the plantbinary vector pCAMBIA1307. The average size of the inserted genesequences was 1.5 kb. The obtained plasmid is shown in FIG. 1.

Example 3: Transformation of the Plants

3.1 Introduction of the FTO Gene of Example 2 into Rice with theTransgenic Technologies

1. Inducing Calluses with Mature Embryos of Rice as Test Materials

1. Sterilization

Mature seeds of rice (japonica Nipponbare) were artificially unshelled.Seeds which were full, bright and clean, and bacterial plaque free wereselected and placed into a 100 ml sterilized flask. 70% alcohol wasadded into the flask to sterilize for 2 minutes. Then, the alcohol wasdecanted, and 20% NaClO was added to soak for 30 minutes. Then, theNaClO was decanted, and the seeds were rinsed 4-5 times with sterilizeddistilled water. Lastly, the seeds were soaked with sterilized distilledwater for 30 minutes.

2. Introduction Culture (Under Aseptic Conditions):

The sterilized seeds were placed on sterilized filter paper. After thewater on the surface were absorbed by the filter paper, the seeds wereplaced into NB medium (PH5.8) containing 2.0 mg/L 2, 4-D at the densityof 12-14 seeds per petri dish. To ensure good induction rate, thegermination orientation of the seeds was made parallel to the medium orslightly downward, and not upward or vertically upward. Then, the petridish was sealed with membrane, and was induced and incubated for 20-30days in an incubator with light at 30° C., 50% humidity until obviouslyloose calluses appeared. Then, subculture was performed.

3. Subculture (Under Aseptic Conditions):

The petri dish was opened on a super-clean operating desk. Calluseswhich naturally divided, grew vigorously and which were hard and brightyellow and which had the diameter of about 3 mm were placed in NB medium(PH5.8) containing 2.0 mg/L 2,4-D and 0.5 mg/L 6-BA at the density of 10calluses per petri dish, and were dark incubated at 30° C. If thecalluses seriously turned soft, they were moved into light to besubcultured. The subculture was performed twice, each 10-15 days (thetime of the next subculture was determined according to how much thecalluses grew).

II. The Culture of the Agrobacterium

The pCAMBIA1307 vector carrying FTO gene and Hygromycin resistance geneshown in FIG. 1 was transfected into Agrobacterium LBA4404. Then,Agrobacterium LBA4404 was seeded into YEP solid medium containing 20mg/L Rifampin (Rif) and SOmg/L kanamycin (Kan). After cultured at 28° C.for two days, Agrobacterium monoclones were picked up to be subject tocolony PCR to test whether the FTO was transferred into theAgrobacterium. Positive monoclones were selected and placed in 4 ml YEP(containing 50 mg/L Kan and 20 mg/L Rif) broth, and wereshaking-cultured at 28° C., 220 rpm for 20-36 h until the OD₆₀₀ of thebacterial solution was 0.8-1.0.

III. Infection and Co-Culture

-   1. The solution of the cultured Agrobacterium was centrifuged at 4°    C., 4000 rmp for 10 min, and was made to suspension with AAM broth    containing 100 μmol/L acetosyringone. The final OD₆₀₀ of the    bacterial solution was about 0.2.-   2. The calluses with certain size was picked up and placed into the    suspension of the Agrobacterium to be infected for 20-30 min.-   3. The calluses were took out and placed on sterilized filter paper    to be dried for 20-30 min so as to prevent undue injury of the    calluses caused by the over-growth of the Agrobacterium during the    co-culture.-   4. The calluses were placed in NB medium (PH5.2) containing 2.0 mg/L    2,4-D and 100 μmol/L acetosyringone and were cultured at 25° C. for    48-72 h in the dark.

IV. Screening of Calluses with Resistance

The calluses were took out and rinsed with shaking by sterilized waterfor 5-6 times. Then, the calluses were placed on sterilized filter paperto be dried and then placed evenly in NB medium (PH 5.8) containing 50mg/L hygromycin for the first screening. The calluses were cultured inthe dark at 28° C. and when there was growth of mold or Agrobacterium,the calluses were timely moved to a new screening plate.

After screening for about 30 days, new calluses were produced. They weremoved to new NB medium (PH 5.8) containing SOmg/L hygromycin for another7-10 day screening. If the growth was obvious, they were positivecalluses. If there was no growth (even if there was no browning ordeath), they were possibly false positive.

V. Introducing the Differentiation of the Calluses with Resistance andRooting

The vigorously grew, yellow calluses after the second screening (toensure that the calluses used to differentiate were not defective) wereplaced in NB medium (PH 5.8) containing 2.0 mg/L 6-BA, 0.5 mg/L kinetin,and 50 mg/L hygromycin at the density of 2-3 positive clones per bottle.It was enough to place a little of calluses per clone. The placedcalluses should have high quality rather than large number. Aftercultured at 27° C. in the dark for 10 days, they were allowed todifferentiate in the light for 10-20 days. After green leaves appeared,they were allowed to root at the light density of 4000 lux, 14 h/d.

The strong seedlings differentiated from each clone were placed in ½N6medium (PH5.8) containing 0.5 mg/L naphthaleneacetic acid and 50 mg/Lhygromycin to induce rooting at the density of 2-3 seedlings eachbottle. The seedlings were cultivated at 27-30′C in the light at thelight density of 4000 lux, 14 h/d. After 7-10 days, the sealingmembranes were opened to add appropriate amount of water. After 2-3days, the seedlings were transplanted.

VI. Training and Transplantation of the Transgenic Seedlings

The rice seedlings with well differentiated roots, stems and leaves werepicked up (when the seedlings grew to the top of the test tubes, thecovers should be opened timely). The sealing membranes were opened andappropriate amount of distilled water or sterilized water was added (toprevent the growth of bacteria in the medium). The seedlings weretrained for about 3 days to one week. After the agar was rinsed, theseedlings were transplanted into earthen bowls in the greenhouse to growand to be tested.

Example 4 Test of the Transformed Seedlings

-   1. PCR detection: The design of FTO gene primers

Primers for detecting human FTO (hFTO): (SEQ ID NO: 13)hFTO-F: 5′-ATGAAGCGCACCCCGACTG-3′; (SEQ ID NO: 14)hFTO-R: 5′-GGGTTTTGCTTCCAGAAGCTGA-3′;Primers for detecting cattle FTO (cFTO): (SEQ ID NO: 15)cFTO-F: 5′-ATGAAGCGGACCCCGACG-3′; (SEQ ID NO: 16)cFTO-R: 5′-GGGCCTGGTTTCCAGAAGCAG-3′;Primers for detecting pig FTO (pFTO): (SEQ ID NO: 17)pFTO-F: 5′-ATGAAGCGAACCCCAACCGC-3′; (SEQ ID NO: 18)pFTO-R: 5′-GGGTTTGGCTTCCAGAAGCAGAC-3′;Primers for detecting Ostreococcus lucimarinus  FTO (olFTO):(SEQ ID NO: 19) olFTO-F: 5′-ATGTCGCCGTCATCCTCCG-3′; (SEQ ID NO: 20)olFTO-R: 5′-CACTTTGTTTTGCTCETCCTCGAGAAA-3′.

The PCR reaction program was: 35 cycles of 95° C. 5 min, 95° C. 15 s,58° C. 15 s, 72° C. 30 s. Extended at 72° C. for 10 min, stored at 4° C.After the reaction, PCR products were subject to 1% agarose gelelectrophoresis analysis.

-   2. Method for rapidly detecting the transgenic seedlings: the    freshly green leaves of about 1 cm were cut and collected from the    seedlings to be tested (both sides had incisions) and were placed    flatly on the test medium (0.7% agar, 1 ml/L 6-BA, 50 mg/L    hygromycin) and were cultured at 28° C. for 48 h (16 h light/8 h    dark each day). The plants which have freshly green leaves were    positive ones while the leaves of the negative seedlings had plaques    of necrosis.

Example 5 Evaluation of the Effects of FTO Genes from Several Species inRice

The test results of the rice of example 3 are shown in table 1 (thejaponica Nipponbare variety which had not been introduced the FTO genewas the control). The data were obtained from 50 transgenic and controlrice plants in maturation stage. Dry weight was weighed after placed in105° C. oven for 20 mins and 80° C. oven for 20 hours.

TABLE 1 Evaluation of the effects of human, cattle, pig and Ostreococcuslucimarinus FTOs introduced into rice (transgenic plants/control plants)Biomass Biomass Biomass of (fresh (dry the whole weight of weight ofplant the the (seeds plus overground overground The Fresh Dry the partsafter parts after introduced Tiller weight of weight of overgroundremoving removing nucleic acids number the seeds the seeds parts) theseeds) the seeds) encoding the Increased Increased Increased IncreasedIncreased Increased FTOs Plant folds folds folds folds folds folds HumanFTO rice 2.54 4.58 4.15 2.61 2.41 3.87 (SEQ ID NO: 5) Cattle FTO 2.154.15 3.71 2.37 2.22 3.61 (SEQ ID NO: 7) Pig FTO 2.08 4.02 3.62 2.34 2.113.60 (SEQ ID NO: 9) Ostreococcus 1.85 3.78 3.41 2.51 2.15 3.45lucimarinus FTO (SEQ ID NO: 11)

Table 1 shows that after the nucleic acids encoding the FTOs of severalspecies were introduced to rice, the biomass and the yield of seeds wereincreased.

The inventor also tested the codon-optimized nucleic acids encoding theFTOs (SEQ ID NOs:6, 8, 10 and 12) by the same method. The data aresimilar to the above, and thus are not shown herein.

Example 6 Evaluation of the Effect of FTO in a Variety of Plants

A variety of plants were transformed with the nucleic acid encodinghuman FTO (SEQ ID NO:5) to obtain a variety of transgenic plant cellsand plants in which the nucleic acid encoding the FTO were introduced.The methods are as follows:

Genetic Transformation Method of Tobacco

1. Experimental Materials

Materials: Tobacco variety: K326; Agrobacterium: LBA4404; HF (human FTO)gene was cloned into the 35S promoter-driven vector pCAMBIA2300. Thescreened resistance in eukaryotic plants was resistance to Kanamycin.

Chemicals: MS macroelements, MS microelements, MS iron salts, indoleacetic acid (IAA), 6-benzylaminoadenine (6-BA), inositol nicotinate(B₁B6), sucrose, agar, cephalosporin (Cef), carbenicillin (Carb),kanamycin (Kn), gentamicin, rifampicin; MS medium (1 L): macroelements(20×) 50 ml, microelements (100×) 10 ml, Fe²′ (100×) 10 ml, sucrose 30g, agar 8 g. The pH was about 6.0.

Pre-culture medium (1 L): macroelements (20×) 50 ml, microelements(100×) 10 ml, Fe²⁺ (100×) 10 ml, 6-BA (1000×) 2 ml, B₁B₆ (200×) 5 ml,glycine (1000×) 1 ml, agar 8 g. The pH was about 6.0. After hightemperature sterilization, IAA (0.2 mg/L) 1 ml was added.

Differentiation culture medium (1 L): based on the pre-culture medium,cephalosporin 2 ml, carbenicillin 1 ml and kanamycin 1 ml were added.

Rooting culture medium (1 L): ½MS, IAA 2 mg/L, sucrose 30 g/L, agar 5.8g/L, pH=5.8

LB broth (1 L): tryptone 10 g, yeast extract 5 g, NaCl 10 g.

MS₀ medium: MS culture medium without the addition of agar and onlycontaining macroelements

2. Tobacco Transformation (Leaf Disc Method)

Tobacco was transformed by leaf disc method. The cut tobacco leaf discswere placed on the pre-culture medium for 1-2 days, and then soaked inAgrobacterium suspension (MS₀ suspended, diluted by 50-100 times) for3-5 minutes. Then, the discs were taken out, and sterile filter paperwas used to absorb the liquid on their surfaces. The infected leaf discswere respectively inoculated on a pre-culture medium covered with twolayers of filter paper and cultured at 26° C. for 20-24 h in dark. Thediscs were rinsed with the sterilized water added with cephalosporin andcarbenicillin (1000 times the mother liquor) for 10 min. The, they werefinally rinsed with sterilized water for 10 min. Subsequently, sterilefilter paper was used to absorb the liquid on their surfaces, and thenthe discs were placed in the differentiation culture medium fordifferentiation culture. In the early stages, subculturing was performedevery 2-3 says, and each subculturing shall be carried out under asepticconditions; after the continual subculturing for three times,subculturing was performed every two weeks. Co-culture was carried outuntil the first bud appeared. The buds were transferred into tissueculture vessels for culturing. When the buds grew to 2 cm, all thecalluses at basal parts of buds and basal leaves were cut on asuper-clean operating desk, and then the buds were placed in the rootingculture medium. When the roots grew to 3 cm, sterile seedlings weretaken out; the solid culture medium was smashed gently, while theresidual culture medium was rinsed off. Thereafter, the sterileseedlings were placed in soil, covered with clear plastic bags(pricked), and cultured for about one week, and then transferredoutdoors (growing in dark in the first 3 days).

Transformation Method of Potato

1. Materials:

Potato variety: Dongnong 303; Agrobacterium: LBA4404; HF (human FTO)gene was cloned into the 35S promoter-driven vector pCAMBIA2300. Thescreened resistance in eukaryotic plants was resistance to Kanamycin.

2. Potato Transformation

The potatoes were cleaned by rinsing with distilled water, soaked with75% alcohol for 30 sec, soaked with 0.1% mercuric chloride for 10 min,cleaned with sterilized water for 5 times, and then peeled to be cutinto slices with the thicknesses around 1 mm. The slices were mixed withthe Agrobacterium solution under gentle shaking to enable theAgrobacterium solution to contact with the explants sufficiently.Sterile filter paper was used to absorb the redundant Agrobacteriumsolution. Then the potato pieces were placed in the co-culture mediumfor dark culture. After the completion of co-culture, the potato pieceswere cleaned for 3 times with sterilized water and liquid MS culturemedium respectively. The redundant Agrobacterium solution was rinsedoff. The potato pieces were transferred into the regeneration culturemedium. Then, the potato pieces were transferred into the rootingculture medium till the buds grew to 1.5-2.0 mm and were cut, forrooting screening propagation.

Genetic Transformation of Soybean

1. Materials

Agrobacterium strain was LBA4404; somatic cell embryo masses of soybeanDongnong L13 at the globular stage; the transgenic plasmid was a 35Spromoter-driven vector pCAMBIA2300 into which the HF gene was cloned.The screened resistance in eukaryotic plants was resistance tokanamycin.

Plant Tissue Culture Medium and Culture Conditions:

Subculture medium: MS+15 mg/L)−20 mg/L 2,4-D+0.8% agar+3% sucrose pH=5.8natural light

Re-suspension culture medium: subculture medium (liquid)+AS (0-100μmol/L) pH=5.8 Natural light

Co-culture medium: infection culture medium+AS (0-100 μmol/L) pH=5.6

Sterilization screening culture medium: subculture medium+(50-300 mg/L)cephalosporin+25-50 mg/L kanamycin

Germination culture medium 1: M S+1% activated carbon+0.8% agar+10%sucrose illumination 16 h/d

Germination culture medium 2: MS+0.8% agar+10% sucrose illumination 16h/d seedling strengthening culture medium: MSB+0.8% agar+3% glucose pH=7illumination 16 h/d

(into the last three culture media, Kanamycin 0-50 mg/L L andcephalosporin 0-300 mg/L were added at the same time)

2. Agrobacterium Infected Transformation and Plant Regeneration

The somatic cell embryo masses of soybean with the diameters around 3 mmwere placed in the prepared Agrobacterium infection liquor forinfection. Then, the bacterial solution was poured away, and the sterilefilter paper was used to absorb the redundant liquid to dry the embryomasses. Thereafter, the embryo masses were inoculated onto theco-culture medium, then onto the sterilization screening culture mediumcontaining 50 mg/L of Kanamycin and 300 mg/L of cephalosporin (theconcentrations decreased in sequence depending upon the situation, withthe proviso that no bacteria grew). The infection time was 10 min. Theco-culture time was 2 to 3 days. Acetosyringone was 100 μmol/L. TheAgrobacterium concentration OD600 was 0.5-0.7. Subculturing wasperformed every 15 to 20 days. The number of the resistant somatic cellembryo masses (with the diameters around 3 mm) as generated wasinspected within 3 months. The transformation ratio (the number of theresistant somatic cell embryo masses/the number of the inoculatedsomatic cell embryo masses×100) was calculated. Then, the resistantsomatic cell embryo masses were transferred into the germination culturemedium 1, and then into the germination culture medium 2 after 20 daysof germination. Till the regenerated resistant small plants were grown,said plants were transferred into the seedling strengthening culturemedium to obtain the transformed plants.

Genetic Transformation of Alfalfa

1. Materials

Alfalfa variety; Gongnon-1; Agrobacterium: LBA4404; a plant binaryexpression vector pCAMBIA2300 into which the HF gene was cloned andresistant to Kanamycin.

The culture medium was a MS culture medium added with various growthregulating substances, with a pH value of about 5.8. The culturetemperature was 23 to 25° C. The illumination intensity was 2 000 lx;the illumination time was 18 h/d. The subculturing was performed aboutevery 20 d.

2. Preparation of Calluses

The hypocotyls were used as explants to undergo callus induction, andsomatic embryo differentiation. After the somatic embryos were mature,somatic embryos were transferred into MS₀ for germination test.

3. Suspending Culture of Embryogenic Calluses

3 g of calluses were inoculated into a 150 mL erlenmeyer flaskcontaining 40 mL of suspending culture solution for performingsuspending culturing. A fresh culture medium was used for change every 7d. The rotational speed of the shaker was 150 r/min. Natural scatteringlight was used for illumination. After 15 d, the cell growth speed wasdetermined. The suitable calluses were inoculated into the bestembryogenic callus induction broth that had been screened out. Therotational speed of the shaker was set as 120 r/min. A fresh culturemedium was used for change every 7 d. The illumination with naturalscattering light lasted about 30 d.

4. Agrobacterium Transformation

Embryogenic calluses were placed in a 2.0 mL small centrifuge tubecontaining 600 μL of liquid co-culture medium for the ultrasonic wavetreatment of 8 s with a parameter of 100 m Hz and the subsequentco-culture of 4 d. The infected suspending embryogenic calluses wereplaced flatwise in a semi-solid co-culture medium containing 100 μmol/Lof acetosyringone for dark culture of 4 d.

5. Screening and Regeneration of Transformed Plants

The co-cultured embryogenic calluses were inoculated into thedifferentiation culture medium for culturing of 50 to 60 d. After 4embryonic stages, embryogenic calluses grew into mature somatic embryos.Then, they were transferred into a somatic embryo germination culturemedium. After about 20 to 30 d, when the plants grew to about 10 cm, theseedlings were transplanted, wherein the used Kanamycin screeningconcentration was 30 mg/L.

Genetic transformation of Rape

1. Materials

Cabbage type rape variety CY2 for genetic transformation; Agrobacteriumstrain EHA105; a plant binary expression vector pCAMBIA1301 into whichthe HF gene was cloned.

2. Agrobacterium Transformation

Fresh bacterial colonies were selected by toothpick to be cultured in aYEB broth containing Km 50 mg/L and Rif 50 mg/L under shaking at 28° C.,till logarithm dividing middle stage (OD600=0.3). The bacterial solutionwas taken for centrifugation at 12 000 r/min for 1 min, washed once withMS culture medium under centrifugation, and then diluted by 10 times.The recipient parent CY2 seeds were sterilized and inoculated onto a ½MS culture medium for culturing sterile seedlings. After 5-7 d,hypocotyls were cut into small sections about 1 cm long to serve asexplants for the Agrobacterium tumefaciens mediated transformation withthe plasmid pCAMBIA1301 carrying HF gene. The transformation was carriedout following the method of Wang Fulin et al. (Journal of NuclearAgricultural Sciences, 5(26): 1129-1134(2011)). Transformants werescreened by hygromycin (Hyg) 10 mg/L. The screened Hyg resistantseedlings rooted, and were domesticated in greenhouse pots for a periodof time, and then transplanted into fields.

Genetic Transformation of Cotton

I. Materials and Reagents

Cotton variety: Zhong 521; Agrobacterium strain GV3101; a plant binaryexpression vector pCAMBIA2300 into which the HF gene was cloned andresistant to Kanamycin.

MBS culture medium: MS inorganic ingredient+B5 organic ingredient as thebasic culture medium, added with different hormone ingredients;

Infection liquor. MS inorganic salts+B5 organics+0.1 mg/L 2,4-D+0.1 mg/LKT+100 μmolVL AS (acetosyringone)+30 g/L glucose;

Co-culture: MS inorganic salts+B5 organics+0.1 mg/L 2,4-D+0.1 mg/LKT+100 μmol/L AS (acetosyringone)+30 g/L glucose+2.5 g/L plant gel;

Screening culture medium: MS inorganic salts+B5 organics+0.1 mg/L2,4-D+0.1 mg/L KT+30 g/L glucose+2.5 g/L plant gel+Kanamycin 50 mg/L+150mg/L carbenicillin

2. Culturing of Explants

The cotton seeds delinted with concentrated sulfuric acid were cleanedwith tap water; seed kernels were taken out. Then, the delinted seedswere placed into a sterilized erlenmeyer flask on a super-cleanoperating desk. The seeds were soaked with 70% to 75% alcohol for 1 min,rinsed with sterilized water, and then soaked with 2% sodiumhypochlorite for 60 min. Thereafter, sodium hypochlorite was pouredaway, and the seeds were rinsed with sterilized water for many times,and soaked in sterilized water for 24 h. After the seeds were revealed,the seed coats were peeled off. The seeds were inoculated into a ½ MSseedling culture medium for dark culture at 28° C. The seedlings at theage of 5 d or so were taken to be cut into small pieces of 0.5-0.6 cm toserve as explants.

3. Co-Culture of Explants and Agrobacterium

Hypocotyls of sterile seedlings were cut into small sections of 0.5 cmor so, and soaked in Agrobacterium solution for 30 to 40 min. Sterilefilter paper was used to absorb the bacterial solution. Said smallsections were placed on a MSB solid culture medium for co-culture of 48h.

4. Induction and Selection of Calluses and Plant Regeneration

The co-cultured hypocotyl cuts were transferred into the MSB solidscreening culture medium containing 50 mg/L of kanamycin and 500 mg/L ofcarbenicillin. Till the culture lasted 60 d, the positive callus massesresistant to Kanamycin were selected to be transferred into the aboveculture medium free of antibiotic for subculturing. Thereafter,according to the state of calluses, particulate embryogenic calluseswere obtained by adjusting the hormone concentration in the culturemedium, and then embryoids were differentiated to culture regeneratedplants.

Genetic Transformation of Wheat (Embryogenic Calluses)

1. Test Materials

Immature embryo tissues were used for culturing. The test variety wasHenong 827. Agrobacterium strain C58. A plant binary expression vectorpCAMBIA2300 into which the HF gene was cloned and resistant toKanamycin.

The culture media used in the genetic transformation process of wheatincluded:

Callus induction culture medium MSWO: MS basic medium+2 mg/L 2,4-D+4mg/L picloride+0.5 g/L glutamine+0.75 g/L MgCl₂+0.1 g/L hydrolyzedcasein+1.95 g/L MES+100 mg/L ascorbic acid+40 g/L maltose+4.5 g/L agar,pH 5.8;

Infection liquor PCM: MS basic medium+2 mg/L 2,4-D+4 mg/L picloride+0.5g/L glutamine+0.75 g/L MgCl₂+0.1 g/L hydrolyzed casein+1.95 g/L MES+100mg/L ascorbic acid+200 μmol/L acetosyringone+40 g/L maltose+4.5 g/Lagar, pH 5.8;

Callus screening culture medium SM: MS basic medium+2 mg/L 2,4-D+4 mg/Lpicloride+0.5 g/L glutamine+0.75 g/L MgCl₂+0.1 g/L hydrolyzedcasein+1.95 g/L MES+100 mg/L ascorbic acid+250 mg/L carbenicillin+25mg/L G418+40 g/L maltose+4.5 g/L agar, pH 5.8;

Resistant callus differentiation culture medium RSM: MS basic medium+2mg/L 2,4-D+4 mg/L picloride+0.5 g/L glutamine+0.75 g/L MgCl₂+0.1 g/Lhydrolyzed casein+1.95 g/L MES+100 mg/L ascorbic acid+250 mg/Lcarbenicillin+25 mg/L G418+0.5 mg/L kinetin+0.2 mg/L naphthalene aceticacid+40 g/L maltose+4.5 g/L agar, pH 5.8;

2. Sterilization of Immature Grains and Inoculation of Immature Embryos

The immature grains were taken 12 to 15 days after pollination of wheatto be surface-sterilized with 70% alcohol for 30 s, sterilized with 0.1%mercuric chloride for 8 min, and cleaned with sterilized water for 4-5times. Immature embryos were picked out with dissecting needles, andwere respectively inoculated onto the callus induction culture mediumMSWO with scutum upwards, for dark culture of 2 weeks at 25° C., andthen transferred into the differentiation culture medium forillumination of 16 h at 25° C. and dark culture of 8 h for 4-6 weeks.

3. Agrobacterium-Mediated Genetic Transformation Procedures

The Agrobacterium C58 containing HF was inoculated uniformly withapplicator onto the LB solid culture medium (pH 7.0, containing 50 mg/Lof kanamycin and 50 mg/L of rifampicin) for culturing of 3 d at 28° C.and then culturing of 1 d at 23° C. Thereafter, a minor amount ofAgrobacterium was scraped from the culture medium to be transferred andinoculated into the YEP broth containing the aforesaid antibiotic forovernight culture at 28° C., at the shaker rotational speed of 250r/min. Agrobacterium was collected till OD600 reached 1.0, and wasre-suspended with PCM infection liquor. The re-suspension was used tosoak calluses for 3 h. Then, the bacterial solution was poured away. Thecalluses were transferred into the culture dish spread with sterilefilter paper for co-culture of 3 d, then transferred into the screeningculture medium for dark culture of 2 weeks at 25° C., and thentransferred into the differentiation culture medium for illuminationculture at 25° C.

Genetic Transformation of Millet

The same method as wheat was used.

Genetic Transformation of Flax

1. Materials

Flax: Heiya 7; Agrobacterium tumefaciens strain EHA105; a plant binaryexpression vector pCAMBIA1301 into which the HF gene was cloned andresistant to Kanamycin.

2. Preparation of Explants

Filled and shiny seeds Heiya 7 were selected, soaked with 75% alcoholfor 5 min, soaked with 20% bleach powder supernatant for 20 min, rinsedwith sterilized water for three times, and then inoculated onto the MSculture medium for dark culture of 5-7 d at 25° C. 2 days beforeapplication, the seeds were placed under illumination for 16 h each dayat 22° C. for use.

3. Preparation of Agrobacterium Bacterial Solution

The propagation of strain EHA105 containing the gene of interest wasperformed using a YEP culture medium, peptone 10 g/L, yeast extract 10g/L, NaCl 5 g/L, and kanamycin 50 mg/L. After inoculation, shakingculture was carried at 28° C. for 2 d. Top phase was removed bycentrifugation for 10 min at 3000 r/min. The bacteria was suspended witha ½ MS broth (OD600=0.5) for transformation.

4. Selective Pressure Test

The sterile flax hypocotyls were cut into small pieces of 0.3-0.5 mm.Said small pieces were soaked with sterilized water for 10 min, absorbeddry by sterile filter paper, and respectively inoculated onto the MSculture medium of kanamycin 50 mg/L for culture at 24-26° C. Theillumination period was 16 h each day.

5. Co-Culture

The flax hypocotyls were cut into small pieces of 0.3-0.5 mm. Said smallpieces were soaked with Agrobacterium EHA105 suspension for 10-20 min,absorbed dry by sterile filter paper, and respectively inoculated ontothe MS, B5 or N6 culture medium of KT 2 mg/L, IAA 3.5 mg/L or HL 150mg/L. The culture conditions were the same as above.

6. Screening Culture and Rooting

The screening culture medium was the same as that for co-culture, andonly differed in the addition of 50 mg/L of kanamycin and 1000 mg/L ofcephalosporin. The explants co-cultured for 3 days with Agrobacteriumwere soaked with 2000 mg/L of cephalosporin solution for 10-20 min,absorbed dry by sterile filter paper, and then inoculated onto thescreening culture medium for culture under the same conditions as shownabove. The sterilization and callus formation were inspected one weekand two weeks after inoculation respectively. The selected resistantbuds were transferred onto the rooting culture medium for inducedrooting.

Genetic transformation of Sunflower

1. Materials

Helianthusannuus Xinkuiza 6; Agrobacterium tumefaciens strain EHA105; aplant expression vector pCAMBIA1301 into which the HF gene was cloned.

2. Strain Culturing

Fresh Agrobacterium tumefaciens single colonies were selected toinoculate into a YEP (1% yeast extract+1% tryptone+0.5% beef extract)broth under shaking at 28° C. overnight. On the next day, they weretransferred and inoculated into a 20 mL YEP broth containing antibioticat 1% inoculation amount for the further vibration and culture tilllogarithm growth period; bacteria were collected by centrifugation anddiluted by MES liquid till OD₆₀₀ reached 0.8 as the workingconcentration for use.

3. Culture Media

MS₀ medium: MS basic ingredients+2% sucrose+0.8% agar, pH 5.8;

GBA basal medium: MS₀+0.5 mg/L BA P+0.25 mg/L IAA+0.1 mg/L GA₃+30 g/Lsucrose+0.8% agar, pH 5.8;

Co-culture medium (M C): GBA+acetosyringone (A CS) (100 mol/L)+30 g/Lsucrose+0.8% agar, pH 5.8;

Screening culture medium (M B): GBA+Carb 400 mg/L+hygromycin 10 mg/L+30g/L sucrose+0.8% agar, pH 5.8;

Rooting medium (M R): ½M S₀+0.2 mg/L N A A+250 mg/L Carb+5 mg/Lhygromycin+30 g/L sucrose+0.8% agar, pH 5.8.

4. Preparation of Explants

The seeds that were filled, uniform in size and had no pests anddiseases were selected, decorticated, soaked with 70% ethanol for 1 min,rinsed with sterilized water twice, sterilized with 1% AgNO₃ for 3 min,and rinsed with sterilized water for three times. The seeds were sowedonto a MS₀ solid culture medium and sprouted in dark at 28° C.; sterileseedlings were obtained after culture for 36-48 h. Roots, cotyledons andphyllopodiums of sterile seedlings were cut to reveal stem tips, andthen cut longitudinally. The obtained explants contained a half of stemtip meristems and two halves of cotyledon axillary buds.

5. Infection

The prepared stem tip explants were soaked in bacterial solutionsufficiently for 10 min, and then taken out. The sterile filter paperwas used to absorb sufficiently the redundant bacterial solution on thesurfaces of explants. The explants were placed onto a MC co-culturemedium for co-culture of 3 d in dark at 28° C., and control wasprovided.

6. Transformant Screening and Plant Regeneration

The explants co-cultured for 3 d were transferred onto a screeningculture medium MB containing 10 mg/mL hygromycin (Hyg) for culture of 2weeks, and then screened for 2-3 turns (2 weeks for each turn). Theselected resistant buds were transferred onto the rooting culture mediumMR for induced rooting.

Genetic Transformation of Taraxacum kok-Saghyz Rodin (Also CalledRussian Dandelion)

1. Materials

Taraxacum kok-saghyz Rodin; Agrobacterium strain: GV3101; plasmid(pCAMBIA2300-35S-HF) which was a binary vector pCAMBIA2300 carrying theHF gene and resistant to Kanamycin.

2. Genetic Transformation and Regeneration of Taraxacum kok-Saghyz Rodin

-   (1) Tissue-cultured seedlings growing well were selected. The edges    of the leaves were removed. The stem of Taraxacum kok-saghyz Rodin    was cut into a length of 2 cm. The leaves were cut into the size of    1 cm². They were placed onto a MS culture medium added with plant    hormones 6-BA and NAA for dark culture of 2 d;-   (2) 200 μl was taken from Agrobacterium GV3101 glycerin tube stored    at −70° C. and containing plasmid pCAMBIA2300-35S-HF to be    inoculated into 50 ml of LB (50 mg/L Gen+100 mg/L Rif+50 mg/L Kan)    liquid for culture overnight;-   (3) The bacterial solution cultured overnight was streaked on the LB    (50 mg/L Gen+100 mg/L Rif+50 mg/L Kan) solid culture medium, placed    upside down, for culture of 2 d at 28° C.;-   (4) Monoclonal colonies were selected for inoculation onto the LB    (50 mg/L Gen+100 mg/L Rif+50 mg/L Kan) broth, for shaking culture of    2 d at 28° C.;-   (5) The cultured bacterial solutions were respectively inoculated    into 100 ml of LB (50 mg/L Gen+100 mg/L Rif+50 mg/L Kan) liquid at a    ratio of 1:100 for enlarged culture, and activated till OD260 of    about 0.6 for infection;-   (6) The aforesaid two bacterial solutions were respectively placed    into 2 50 ml sterile large centrifuge tubes, and centrifuged for 10    min at 5000 rpm;-   (7) The supernatant was discarded. The bacteria was suspended by 100    ml of MS broth, and cultured for 5 min at 28° C.;-   (8) The above explants cultured in dark for 2 d were placed into the    selected bacterial solution for shaking culture of 20 min at 28° C.;-   (9) The infected explants were spread on the sterile dry filter    paper, with the redundant bacterial solution absorbed, for dark    culture of 2 d;-   (10) After 2 d, the explants were taken out and placed onto the MS    (1 mg/L 6-BA+0.1 mg/L NAA+400 mg/L Cb (carbenicillin)+50 mg/L Kan    (Kanamycin)) solid culture medium; the plate was poured every half a    month;-   (11) When adventitious shoots grew to 2 cm, single shoots were    broken off and inserted into the rooting culture medium ½MS (0.2    mg/L NAA+50 mg/L Kan+400 mg/L Cb) for growing;-   (12) After one month, the strong tissue-cultured seedlings were    domesticated for 1 d, transplanted into nutrient soil (turfy    soil:vermiculite=3:1), covered with film for 1 week, and placed in    cultivation room for subsequent culturing.

Genetic Transformation of Maize

1. Materials and Reagents

Maize variety: Qi 319; Agrobacterium strain: GV3101; plasmid(pCAMBIA2300-35S-HF) which was a binary vector pCAMBIA2300 carrying theHF gene and resistant to Kanamycin.

D-culture medium: NaFeEDTA 10 ml/L+N6 macroelements 50 ml/L+B5microelements 10 ml/L+Di comba (2,4-D) 1 ml/L (5 ml/L)+RTV 10ml/L+Casamina acids (casein hydrolase) 0.5 g/L+L-Prine 700 mg/L+inositol100 mg/L+Sucrose 20 g/L, pH 5.8

D-Inf culture medium: NaFeEDTA 10 ml/L+N6 macroelements 50 ml/L+B5microelements 10 ml/L+Di comba (2,4-D) 1 ml/L+RTV 10 ml/L+Casamina acids(casein hydrolase) 0.5 g/L+L-Prine 700 mg/L+inositol 100 mg/L+Sucrose68.5 g/L+glucose 36 g/L, pH 5.2

D-AS culture medium: D-culture medium+glucose 10 g/L+agar powder 8g/L+AS (0.5 M) 200 μl+AgNO₃ 1 ml/L (both AS and AgNO₃ had the finalconcentration of 100 μM, and were added after sterilization of theculture medium when being slightly cool for uniform mixing)

D-Cefculture medium: D-culture medium+1 ml/L AgNO₃+cef 1 ml/L

2. Genetic Transformation and Regeneration of Maize

-   (1) Preparation of maize immature embryo: maize leaves, maize silks    and some redundant parts were peeled off; a knife was inserted into    the upper portion for adding 70% ethanol; the maize entered the    super-clean operating desk; 30 s later, it was taken out and blown    dry on the super-clean operating desk for about 15-20 min; ⅔ of the    surface of the grain was peeled off; immature embryo was peeled out.-   (2) The bacterial solution added into D-Inf (containing AS) was    diluted till OD600 of 0.3-0.5 and stood for more than 1 h. The    immature embryo was washed once with D-Inf (free of AS), and then    soaked into the bacterial solution, upside down with hand for 30 s,    and stood for 5 min. Observed with no apparent wounds, the immature    embryo was taken out, absorbed dry with sterile filter paper, and    placed onto the D-AS solid culture medium for co-culture of 3 d in    dark at 25° C.-   (3) Stage of restoring culture: the immature embryo co-cultured for    3 d was rinsed in sterilized water (added with 1‰ of cef) for three    times, 20 min for each time, and then absorbed dry with filter    paper, and transferred onto the D-Cef solid culture medium, and    restoring cultured in dark at 25° C. for 7 d. Thereafter,    pressurization screening stage was performed: in 4 cycles, with the    interval of 2 weeks between any two.

The 1^(st) cycle: D culture medium+cef (1‰)+PPT (5 mg/ml)

The 2^(nd) cycle: D culture medium+cef (1‰)+PPT (10 mg/ml)

The 3^(rd) cycle: D culture medium+cef (1‰)+PPT (10 mg/ml)

The 4^(th) cycle: D culture medium+cef (1‰)+PPT (10 mg/ml)

-   (4) Restoration stage after screening: D culture medium+6-BA (5    mg/L), wherein 2,4-D or Dicamba concentration was diluted by 5    times, and sucrose was 30 g/L (or sucrose 20 g/L, glucose 10 g/L);    the period was 2 weeks; dark culture.-   (5) Induction stage after screening: D culture medium+6-BA (5 mg/L),    wherein 2,4-D or Dicamba concentration was diluted by 5 times, and    sucrose was 50 g/L, without the addition of glucose, +cef 1 ml/L;    dark culture.-   (6) Differentiation stage: D culture medium, yet without the    addition of any hormone; sucrose concentration was 30 mg/L, without    the addition of glucose, +cef 1 ml/L; illumination culture.-   (7) Rooting stage: ½ MS culture medium.

Genetic Transformation of False Flax (Camelia saiva) (by a FlowerDripping Method)

1. Materials and Reagents

Camelina sativa; Agrobacterium strain: GV3101; plasmid(pCAMBIA2300-35S-HF) which was a binary vector pCAMBIA2300 carrying theHF gene and resistant to Kanamycin.

2. Genetic Transformation of Camelina sativa by a Flower Dripping Method

-   (1) Wild-type Camelina sativa was sowed in nutrient soil    (humus:vermiculite:perlite=4:2:1), 5 grains/pot (diameter: 9 cm),    and placed in cultivation room for culturing. The cultivation room    had the temperature of 16 to 20° C. and the relative humidity of    60%. The illumination intensity was 4 000 lx. The illumination    period was 16 h illumination/8 h dark. When the main stem of    Camelina sativa plant grew to 5 cm and the lateral branches were    just bud-like, the top inflorescences were removed to promote the    growth and development of secondary branches. When the plants grew    to the full-bloom stage, they were prepared for transformation. 2 d    before transformation, the formed fruit pods were cut off.-   (2) When Agrobacterium GV3101 containing the plasmid    pCAMBIA2300-35S-HF was cultured till the OD600 value was 0.8, it was    infiltrated isometrically into the culture medium (½ MS, 5% sucrose,    200 μL/L Silwet L-77) to re-suspend bacteria and transform Camelina    sativa. Upon transformation, a pipettor was used to suck    Agrobacterium re-suspension for infiltration into the culture medium    and dripping onto the flower buds of Camelina sativa, ensuring that    each flower bud was dripped. Thereafter, preservative films were    used to coat the transformed plants to maintain humidity. The plants    stood upright in dark in the cultivation room for culture of 1 d,    and then were cultured under normal conditions.-   (3) After the plants were mature, the seeds were collected and    designated as TO generation seeds. 50 mg/L of kanamycin was used to    screen the obtained TO generation Camelina sativa seeds to obtain    positive transgenic plants.

The yield and biomass of the transgenic plants comprising the nucleicacid encoding the FTO obtained from the above methods and the controlplants that do not comprise FTO were measured. The results are shown intable 2.

TABLE 2 Changes of the yield and biomass after the nucleic acid encodingthe FTO were introduced into a variety of plants Biomass (fresh weightof the overground parts Yield after removing Plant (Organs forevaluating the Increased the seeds) gene yield) folds Increased foldsHuman maize (seed) 4.12 2.81 FTO soybean (seed) 4.00 2.75 (SEQ tobacco(leaf) 3.65 2.54 ID potato (tuber) 3.83 2.61 NO: 5) alfalfa (stem andleaf) 2.43 2.43 rape (seed) 4.21 2.94 Russian dandelion (Taraxacum 3.252.88 kok-saghyz) (rubber solution in the root) cotton (seed cotton) 3.912.67 wheat 3.78 2.58 millet (seed) 4.08 2.79 flax (for oil) (seed) 3.772.58 sunflower (seed) 3.88 2.69 false flax (seed) 4.09 2.79

From table 2, it can be seen that after FTO was introduced into avariety of plants, the yield and the biomass were increased.

1. A transgenic plant in which a nucleic acid molecule encoding an m⁶Ademethylase is introduced, wherein said m⁶A demethylase has thefollowing two domains: i) N-terminal domain (NTD) having the function ofAlkB oxidation demethylase; and ii) C-terminal domain (CTD).
 2. Thetransgenic plant of claim 1, wherein said m⁶A demethylase is FTO (fatmass and obesity-associated) protein.
 3. The transgenic plant of claim2, wherein said FTO protein is from vertebrates or marine algae.
 4. Thetransgenic plant of claim 3, wherein said FTO protein has at least 40%,preferably at least 50%, more preferably at least 60%, more preferablyat least 70%, more preferably at least 80%, more preferably at least90%, more preferably at least 95%, more preferably at least 99%, mostpreferably 100% identity to any one of SEQ ID NOs:1-4.
 5. The transgenicplant of claim 1, wherein said nucleic acid molecule encoding the m⁶Ademethylase has at least 90%, preferably at least 95%, more preferablyat least 99%, most preferably 100% identity to any one of SEQ IDNOs:5-12.
 6. The transgenic plant of claim 1, wherein said plantexhibits an increased biomass, an increased yield or the combinationthereof when compared with a control plant which does not comprise thenucleic acid molecule encoding the m⁶A demethylase.
 7. The transgenicplant of claim 1, wherein said plant is selected from the groupconsisting of rice, maize (Zea mays), soybean, tobacco, potato, alfalfa(Medicago sativa), rape (Brassica), Russian dandelion (TaraxacumKok-saghyz), cotton, wheat, millet (Panicum miliaceum), flax, sunflowerand false flax (Camelina sativa).
 8. A tissue, an organ, a pollen, aseed, a grain or a fruit of the plant of claim
 1. 9. A progeny plant ofthe plant of claim
 1. 10. A plant cell in which a nucleic acid moleculeencoding an m⁶A demethylase is introduced, wherein said m⁶A demethylasehas the following two domains: i) N-terminal domain (NTD) having thefunction of AlkB oxidation demethylase; and ii) C-terminal domain (CTD).11. The plant cell of claim 10, wherein said m⁶A demethylase is FTOprotein.
 12. The plant cell of claim 11, wherein said FTO protein isfrom vertebrates or marine algae.
 13. The plant cell of claim 12,wherein said FTO protein has at least 40%, preferably at least 50%/o,more preferably at least 60%, more preferably at least 70%, morepreferably at least 80%, more preferably at least 90%, more preferablyat least 95%, more preferably at least 99%, most preferably 100%identity to any one of SEQ ID NOs:1-4.
 14. The plant cell of claim 1,wherein said nucleic acid molecule encoding the m⁶A demethylase has atleast 90%, preferably at least 95%, more preferably at least 99%, mostpreferably 100% identity to any one of SEQ ID NOs:5-12.
 15. A method forproducing a transgenic plant exhibiting an increased biomass, anincreased yield or the combination thereof, wherein said methodcomprises: a) introducing a nucleic acid molecule encoding an m⁶Ademethylase into a regenerable plant cell, wherein said m⁶A demethylasehas the following two domains: i) N-terminal domain (NTD) having thefunction of AlkB oxidation demethylase; and ii) C-terminal domain (CTD);and b) regenerating a transgenic plant from the regenerable plant cell,wherein the transgenic plant comprises in its genome said nucleic acidmolecule encoding the m⁶A demethylase, and exhibits an increasedbiomass, an increased yield or the combination thereof when comparedwith a control plant which does not comprise the nucleic acid moleculeencoding the m⁶A demethylase.
 16. The method of claim 15, wherein saidmethod further comprises: c) obtaining a progeny plant derived from thetransgenic plant of step b), wherein said progeny plant comprises in itsgenome said nucleic acid molecule encoding the m⁶A demethylase, andexhibits an increased biomass, an increased yield or the combinationthereof when compared with a control plant which does not comprise thenucleic acid molecule encoding the m⁶A demethylase.
 17. The method ofclaim 1, wherein said m⁶A demethylase is FTO protein.
 18. The method ofclaim 17, wherein said FTO protein is from vertebrates or marine algae.19. The method of claim 18, wherein said FTO protein has at least 40%,preferably at least 50%, more preferably at least 60%, more preferablyat least 70%, more preferably at least 80%, more preferably at least90%, more preferably at least 95%, more preferably at least 99%, mostpreferably 100% identity to any one of SEQ ID NOs:1-4.
 20. The method ofclaim 1, wherein said nucleic acid molecule encoding the m⁶A demethylasehas at least 90%, preferably at least 95%, more preferably at least 99%,most preferably 100% identity to any one of SEQ ID NOs:5-12.
 21. Themethod of claim 1, wherein said plant is selected from the groupconsisting of rice, maize (Zea mays), soybean, tobacco, potato, alfalfa(Medicago saliva), rape (Brassica), Russian dandelion (TaraxacumKok-saghyz), cotton, wheat, millet (Panicum miliaceum), flax, sunflowerand false flax (Camelina sativa).